HIV-1 mutations selected for by β-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine

ABSTRACT

The present invention discloses a method for treating HIV that includes administering β-D-D4FC or its pharmaceutically acceptable salt or prodrug to a human in need of therapy in combination or alternation with a drug that induces a mutation in HIV-1 at a location other than the 70(K to N), 90 or the 172 codons of the reverse transcriptase region. Also disclosed is a method for using β-D-D4FC as “salvage therapy” to patients which exhibit drug resistance to other anti-HIV agents. β-D-D4FC can be used generally as salvage therapy for any patient which exhibits resistance to a drug that induces a mutation at other than the 70(K to N), 90 or the 172 codons.

This application is a continuation of U.S. application Ser. No.09/677,161, filed on Oct. 2, 2000, now U.S. Pat. No. 6,391,859, which isa continuation of U.S. application Ser. No. 09/488,874, filed on Jan.21, 2000, now abandoned, which claims priority to U.S. ProvisionalApplication Ser. No. 60/116,773, filed on Jan. 22, 1999, now abandoned.

This invention is partially funded by a grant from the United StatesNational Institutes of Health under Grant No. 1R01-A1-41980-01. The U.S.government has certain rights to this invention.

BACKGROUND OF THE INVENTION

In 1983, the etiological cause of AIDS was determined to be the humanimmunodeficiency virus (HIV). In 1985, it was reported that thesynthetic nucleoside 3′-azido-3′-deoxythymidine (AZT) inhibits thereplication of human immunodeficiency virus. Since then, a number ofother synthetic nucleosides, including 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), 2′,3′-dideoxy-2′,3′-didehydrothymidine(D4T), cis-2hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane(FTC), (−)-cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (3TC),have been proven to be effective against HIV. After cellularphosphorylation to the 5′-triphosphate by cellular kinases, thesesynthetic nucleosides are incorporated into a growing strand of viralDNA, causing chain termination due to the absence of the 3′-hydroxylgroup. They can also inhibit the viral enzyme reverse transcriptase.

It has been recognized that drug-resistant variants of HIV can emergeafter prolonged treatment with an antiviral agent. Drug resistance mosttypically occurs by mutation of a gene that encodes for an enzyme usedin viral replication, and most typically in the case of HIV, reversetranscriptase, protease, or DNA polymerase. Recently, it has beendemonstrated that the efficacy of a drug against HIV infection can beprolonged, augmented, or restored by administering the compound incombination or alternation with a second, and perhaps third, antiviralcompound that induces a different mutation from that caused by theprinciple drug. Alternatively, the pharmacokinetics, biodistribution, orother parameter of the drug can be altered by such combination oralternation therapy. In general, combination therapy is typicallypreferred over alternation therapy because it induces multiplesimultaneous pressures on the virus. One cannot predict, however, whatmutations will be induced in the HIV-1 genome by a given drug, whetherthe mutation is permanent or transient, or how an infected cell with amutated HIV-1 sequence will respond to therapy with other agents incombination or alternation. This is exacerbated by the fact that thereis a paucity of data on the kinetics of drug resistance in long-termcell cultures treated with modern antiretroviral agents.

HIV-1 variants resistant to 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (DDI) or 2′,3′-dideoxycytidine (DDC) have beenisolated from patients receiving long term monotherapy with these drugs(Larder B A, Darby G, Richman D D. Science 1989;243:1731–4; St Clair MH, Martin J L, Tudor W G, et al. Science 1991;253:1557–9; St Clair M H,Martin J L, Tudor W G, et al. Science 1991;253:1557–9; and Fitzgibbon JE, Howell R M, Haberzettl C A, Sperber S J, Gocke D J, Dubin D T.Antimicrob Agents Chemother 1992;36:153–7). Mounting clinical evidenceindicates that AZT resistance is a predictor of poor clinical outcome inboth children and adults (Mayers D L. Lecture at the Thirty-secondInterscience Conference on Antimicrobial Agents and Chemotherapy.(Anaheim, Calif. 1992); Tudor-Williams G, St Clair M H, McKinney R E, etal. Lancet 1992;339:15–9; Ogino M T, Dankner W M, Spector S A. J Pediatr1993;123:1–8; Crumpacker C S, D'Aquila R T, Johnson V A, et al. ThirdWorkshop on Viral Resistance. (Gaithersburg, Md. 1993); and Mayers D,and the RV43 Study Group. Third Workshop on Viral Resistance.(Gaithersburg, Md. 1993)). The rapid development of HIV-1 resistance tononnucleoside reverse transcriptase inhibitors (NNRTIs) has also beenreported both in cell culture and in human clinical trials (Nunberg J H,Schleif W A, Boots E J, et al. J Virol 1991 ;65(9):4887–92; Richman D,Shih C K, Lowy I, et al. Proc Natl Acad Sci (USA) 1991;88 :11241–5;Mellors J W, Dutschman G E, Im G J, Tramontano E, Winkler S R, Cheng YC. Mol Pharm 1992;41:446–51; Richman D D and the ACTG 164/168 StudyTeam. Second International HIV-1 Drug Resistance Workshop. (Noordwijk,the Netherlands. 1993); and Saag M S, Emini E A, Laskin O L, et al. NEngl J Med 1993;329:1065–1072). In the case of the NNRTI L'697,661,drug-resistant HIV-1 emerged within 2–6 weeks of initiating therapy inassociation with the return of viremia to pretreatment levels (Saag M S,Emini E A, Laskin O L, et al. N Engl J Med 1993;329:1065–1072).Breakthrough viremia associated with the appearance of drug-resistantstrains has also been noted with other classes of HIV-1 inhibitors,including protease inhibitors (Jacobsen H, Craig C J, Duncan I B,Haenggi M, Yasargil K, Mous J. Third Workshop on Viral Resistance.(Gaithersburg, Md. 1993)). This experience has led to the realizationthat the potential for HIV-1 drug resistance must be assessed early onin the preclinical evaluation of all new therapies for HIV-1.

2′,3′-Dideoxy-2′,3′-didehydro-5-fluorocytidine (D4FC) is a knowncompound. European Patent Application Publication No. 0 409 227 A2 filedby Ajinomoto Co., Inc., discloses β-D-D4FC (Example 2) and its use totreat hepatitis B. Netherlands Patent No. 8901258 filed by StichtingRega V. Z. W. discloses generally5-halogeno-2′,3′-dideoxy-2′,3′-didehydrocytidine derivatives for use intreating HIV and hepatitis B (“HBV”). U.S. Pat. No. 5,703,058 disclosesa method for the treatment of HIV and HBV infection that includesadministering an effective amount of β-L-D4FC in combination oralternation withcis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane,cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane,9-[4-(hydroxymethyl)-2-cyclopenten-1-yl)-guanine (carbovir),9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon,3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine(L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T). U.S. Pat. No.5,905,070 discloses a method for the treatment of HIV and HBV infectionthat includes administering an effective amount of β-D-D4FC incombination or alternation withcis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane,cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane,9-[4-(hydroxymethyl)-2-cyclopenten-1-yl)-guanine (carbovir),9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon,3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine(L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T).

It is an object of the present invention to determine the optimaladministration of β-D-D4FC for the treatment of HIV.

It is another object of the present invention to provide a method andcomposition that includes β-D-D4FC for the treatment of patientsinfected with HIV that exhibits advantageous or improvedpharmacokinetic, biodistribution, metabolic, resistance or otherparameters over administration of β-D-D4FC alone.

It is yet another object of the present invention to provide a methodand composition for the treatment of patients infected with HIV in whichβ-D-D4FC is administered in combination or alternation with a secondcompound that acts synergistically with β-D-D4FC against the virus.

It is still another object of the present invention to provide a methodand composition for the treatment of patients infected with a drugresistant form of HIV.

It is another object of the present invention to provide a method andkit to assess how to best administer β-D-D4FC.

SUMMARY OF THE INVENTION

It has been discovered that β-D-D4FC induces mutations in HIV-1 at the70(K to N), 90 and the 172 codons of the reverse transcriptase region ofthe virus. Based on this discovery, a method for treating HIV isprovided that includes administering β-D-D4FC or its pharmaceuticallyacceptable salt or prodrug to a human in need of therapy in combinationor alternation with a drug that induces a mutation in HIV-1 at alocation other than the 70(K to N), 90 or the 172 codons of the reversetranscriptase region. This invention can be practiced by referring topublished mutation patterns for known anti-HIV drugs, or by determiningthe mutation pattern for a new drug.

Based on this discovery, a method for using β-D-D4FC as “salvagetherapy” to patients which exhibit drug resistance to other anti-HIVagents is also provided. It has been discovered that β-D-D4FC is notsignificantly cross-resistant to AZT, DDC, DDI, D4T, 3TC, (−)-FTC orβ-L-D4FC. In contrast, β-L-D4FC rapidly induces a mutation at codon 184(methionine to valine), resulting in a high level of resistance to 3TCand FTC. β-D-D4FC can be used generally as salvage therapy for anypatient which exhibits resistance to a drug that induces a mutation atother than the 70(K to N), 90 or the 172 codons.

The invention disclosed herein more generally includes at least thefollowing embodiments:

(i) A method for treating an HIV infection in a human comprisingadministering an effective amount of β-D-D4FC or its pharmaceuticallyacceptable prodrug or salt to the human, optionally in apharmaceutically acceptable carrier, in combination or alternation witha drug that induces a mutation in HIV-1 at a location other than the70(K to N), 90 or 172 codon of the reverse transcriptase region, andwhich is other thancis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane,cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane,9-[4-(hydroxymethyl)-2-cyclopenten-1-yl)-guanine (carbovir),9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon,3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine(L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T).

(ii) A method for treating an HIV infection in a human comprisingadministering an effective amount of β-D-D4FC or its pharmaceuticallyacceptable salt to the human, optionally in a pharmaceuticallyacceptable carrier, in combination or alternation with a drug thatinduces a mutation in HIV-1 at codon 70 from K to N (i.e., lysine toasparagine), a mutation at codon 90 from V to I (i.e., valine toisoleucine), or mutation at codon 172 from R to K (i.e., arginine tolysine) of the reverse transcriptase region, and which is other thancis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane,cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathilane,9-[4-(hydroxymethyl)-2-cyclopenten-1-yl)-guanine (carbovir),9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon,3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine(L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T).

(iii) A method for treating a patient infected with a strain of HIVvirus that is resistant to 3TC, comprising administering an effectiveamount of β-D-D4FC or its pharmaceutically acceptable prodrug or salt tothe patient optionally in a pharmaceutically acceptable carrier.

(iv) A method for treating a patient infected with a strain of HIV virusthat is resistant to AZT, comprising administering an effective amountof β-D-D4FC or its pharmaceutically acceptable prodrug or salt to thepatient optionally in a pharmaceutically acceptable carrier.

(v) A method for treating a patient infected with a strain of HIV virusthat is resistant tocis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, comprisingadministering an effective amount of β-D-D4FC or its pharmaceuticallyacceptable prodrug or salt, to the patient optionally in apharmaceutically acceptable carrier.

(vi) A method for treating a patient infected with a strain of HIV virusthat is resistant tocis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane, comprisingadministering an effective amount of β-D-D4FC, or its pharmaceuticallyacceptable prodrug or salt, to the patient optionally in apharmaceutically acceptable carrier.

(vii) A method for treating a patient infected with a strain of HIVvirus that is resistant to 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T),comprising administering an effective amount of β-D-D4FC, or itspharmaceutically acceptable prodrug or salt, to the patient optionallyin a pharmaceutically acceptable carrier.

(viii) A method for treating a patient infected with a strain of HIVvirus that is resistant to 2′,3′-dideoxyinosine (DDI), comprisingadministering an effective amount of β-D-D4FC, or its pharmaceuticallyacceptable prodrug or salt, to the patient optionally in apharmaceutically acceptable carrier.

(ix) A method for treating a patient infected with a strain of HIV virusthat is resistant to 2′,3′-dideoxycytidine (DDC), comprisingadministering an effective amount of β-D-D4FC, or its pharmaceuticallyacceptable prodrug or salt, to the patient optionally in apharmaceutically acceptable carrier.

(x) A method for treating a patient infected with HIV comprisingadministering an effective amount of β-D-D4FC or its prodrug orpharmaceutically acceptable salt in combination or alternation with aneffective amount of(S)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one(SUSTIVA, see U.S. Pat. No. 5,519,021).

The disclosed combination, alternation, or salvage regiments are usefulin the prevention and treatment of HIV infections and other relatedconditions such as AIDS-related complex (ARC), persistent generalizedlymphadenopathy (PGL), AIDS-related neurological conditions, anti-HIVantibody positive and HIV-positive conditions, Kaposi's sarcoma,thrombocytopenia purpurea and opportunistic infections. In addition,these compounds or formulations can be used prophylactically to preventor retard the progression of clinical illness in individuals who areanti-HIV antibody or HIV-antigen positive or who have been exposed toHIV.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing of β-D-D4FC.

FIG. 2 is a graph of the concentration of β-D-D4FC in micromolar versusthe percent inhibition of p24 antigen production. The figure illustratesthe selection of virus with reduced sensitivity to β-D-D4FC.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein, the term “resistant virus” refers to a virus thatexhibits a three, and more typically, five or greater fold increase inEC₅₀ compared to naive virus in a constant cell line, including, but notlimited to peripherial blood mononuclear cells (PBMCs), or MT2 or MT4cells.

The term D-D4FC is used interchangeably with the term β-D-D4FC below.

As used herein, the term “substantially pure” or “substantially in theform of one optical isomer” refers to a nucleoside composition thatincludes at least 95% to 98%, or more, preferably 99% to 100%, of asingle enantiomer of that nucleoside. In a preferred embodiment,β-D-D4FC is administered in substantially pure form for any of thedisclosed indications.

As used herein, the term “prodrug” refers to the 5′ and N⁴ acylated,alkylated, or phosphorylated (including mono, di, and triphosphateesters as well as stabilized phosphates and phospholipid ) derivativesof D-D4FC. In one embodiment, the acyl group is a carboxylic acid esterin which the non-carbonyl moiety of the ester group is selected fromstraight, branched, or cyclic alkyl, alkoxyalkyl includingmethoxymethyl, aralkyl including benzyl, aryloxyalkyl includingphenoxymethyl, aryl including phenyl optionally substituted by halogen,alkyl, alkyl or alkoxy, sulfonate esters such as alkyl or aralkylsulphonyl including methanesulfonyl, trityl or monomethoxytrityl,substituted benzyl, trialkylsilyl, or diphenylmethylsilyl. Aryl groupsin the esters optimally comprise a phenyl group. The alkyl group can bestraight, branched or cyclic and is preferably C₁ to C₁₈.

As used herein, the term “pharmaceutically acceptable salts” refers topharmaceutically acceptable salts which, upon administration to therecipient, are capable of providing directly or indirectly, β-D-D4FC, orthat exhibit activity themselves.

The abbreviations of amino acids used herein are described in Table 1.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic Acid Asp D GAC GAU GAC GAU Glutamic Acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Clycine Gly G GGA GCG GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG GUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAUII. Mutations in HIV-1 Reverse Transcriptase Selected for by β-D-D4FC

Both the D- and L-enantiomers ofβ-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (D4FC) are potent andselective inhibitors of HIV-1, although the D-enantiomer is moreselective. The in vitro development of resistance to D-D4FC was assessedby serial passage of HIV-I_(LA1) in MT-2 cells and peripheral bloodmononuclear cells (PBMC) in the presence of increasing concentrations ofdrug. Variants resistant to D-D4FC arose only after prolonged exposureto the drug. Virus obtained after 20 passages in MT-2 cells exhibited5.3-fold resistance to D-D4FC. Resistant virus could not be isolated inPBMC despite multiple attempts. DNA sequencing of RT from virus selectedin MT-2 cells revealed two mutations: K65R and V179D. The selection ofD-D4FC resistant virus was repeated in MT-2 cells and variantsexhibiting 19.3-fold resistance encoded three novel RT mutations: K70N,V901 and R172K. A K65R recombinant HIV-1_(LA1) virus exhibited 3.9-foldresistance to D-D4FC. VI 79D, a mutation conferring resistance tononnucleoside RT inhibitors, is most likely compensatory for K65R. Therole of the other mutations in resistance to D-D4FC was also evaluatedby construction of recombinant virus with single and multiple mutations,however, none of the recombinants tested demonstrated >2-foldresistance.

Materials and Methods

Chemicals. D-D4FC was synthesized in one of our laboratories asdescribed previously (Shi et al, 1999). It was prepared as 10 mM stocksolutions in sterile water and stored at −20° C. The compound was thawedand diluted to the desired concentration immediately before use.

Cells. MT-2 cells (AIDS Research and Reference Reagent Program, NationalInstitute of Allergy and Infectious Diseases, National Institutes ofHealth, contributed by D. Richman) were cultured in RPMI 1640 (WhittakerM. A., Bioproducts, Walkersville, Md.) supplemented with 10% fetalbovine serum, 10 mM HEPES buffer, penicillin (50 IU/ml) and streptomycin(50 μg/ml).

Viruses. HIV-1_(LA1), a molecularly cloned clinical isolate, was usedboth as the starting virus for the resistance selection as well as forthe generation of recombinant mutants. Stock preparations of HIV-1_(LA1)were prepared by electrophorating 5–10 jig of proviral plasmid DNA into1.3×10⁷ MT-2 cells. At peak viral cytopathic effect (generally 7 dayspost transfection), the supernatant from infected cultures wascollected, aliquoted and stored at −80° C. until use. Virus preparationswere titered by threefold endpoint dilution in MT-2 cells, and theTCID₅₀ was calculated with the Reed and Muench equation.

Selection of resistant virus. Prior to starting the selection of D-D4FCresistant virus, the starting virus (HIV-1_(LA1)) was passaged ascell-free virus for 10 cycles in MT-2 cells in the absence of drug.D-D4FC resistant virus was selected by serially passaging HIV-1_(LA1) inMT-2 cells in the presence of gradually increasing concentrations ofD-D4FC. The selection for D-D4FC resistant virus was conducted twice.Selection was initiated by inoculating 1×10⁶ MT-2 cells with 0.01 mol ofvirus. At peak viral cytopathic effect (4–7 days post infection),supernatant from the infected cultures was collected and 0.1–0.3 ml weresubsequently used to initiate another cycle of infection. Supernatantwas also aliquoted and stored at −80° C. for characterization ofselected virus. Virus was passaged at least three times at eachconcentration, the number of cycles at any given concentration of drugbeing dependent on the ability of virus to grow at the particularconcentration of D-D4FC. During the first selection procedure, virus waspassaged once in the absence of drug prior to increasing the drugconcentration (Table 2). As a control, virus was also passaged inparallel in the absence of drug. The first selection was initiated at0.75 μM and gradually increased to 4.0 μM during the course of 37 cyclesof cell-free passage. The second selection was initiated at 0.2 μM andgradually increased to 6.2 μM during the course of 27 cycles ofcell-free passage (Table 2).

TABLE 2 Selection #1 Selection #2 Passage Passage Number [D-Fd4C] μMNumber [D-Fd4c] μM 1–2 0.75 1–3 0.2 3–4 1.5 4–6 0.4  5 1.0 7–9 0.8  6 010–12 1.6 7–9 1.5 13 3.2 10–12 2.0 14–16 1.6 13–16 3.0 17–19 3.2 17 020–22 4.8 18–20 4.5 23–25 6.2 21 0 26 12.0 22–35 4.5 27 6.2 36–38 1.039–41 2.0 42–43 4.0

Antiviral susceptibility assays. Virus susceptibility to D-D4FC wasmeasured by measuring percent inhibition of p24 antigen production.Briefly, MT-2 cells (1×10⁵ cells/ml) were infected with virus at a moiof 0.01 in the presence of serial D-D4FC dilutions. Each dilution wastested in triplicate. Culture supernatants were harvested day 7 postinfection and assayed for p24 antigen production using a commercialassay (DuPont, NEN Products, Wilmington, Del.). Virus susceptibility isexpressed as the concentration of drug required to inhibit production ofp24 antigen by 50% (EC₅₀).

DNA sequencing of selected virus reverse transcriptase. The viral RNA(vRNA) from selected virus was isolated using TRIzol Reagent (GibcoBRL,Grand Island, N.Y.). The full-length RT coding region was amplified byRT-PCR. The PCR product was subsequently purified using Wizard PCR preps(Promega, Madison, Wis.) and sequenced.

Production of mutant recombinant HIV-1. Mutant RT was generated usingthe Altered SitesII in vitro Mutagenesis System (Promega, Madison,Wis.). Mutagenesis was carried out on HIV-1_(LA1), RT cloned into amutagenesis vector (PALTER, Promega). The presence of the desiredmutation was determined by direct sequencing of the RT gene. The mutantRT was subsequently ligated into the pxxHIV-1_(LA1) vector. Stocks ofmutant virus were then prepared by electrophorating 5–10 μg of DNA into1.3×10⁷ MT-2 cells as described above.

Results and Discussion

Phenotyping of resistant virus. Virus resistant to D-D4FC was selectedafter 37 (selection #1) and 20 (selection #2) cycles of infection inMT-2 cells. Assessment of the D-D4FC susceptibility of virus fromselection #1 passage 37 (p37 D-D4FC#1) demonstrated that p37 D-D4FC#l is19.4-fold less sensitive to D-D4FC than wild type as demonstrated by anincrease in the EC₅₀, from 0.21 μM to 4.07 μM (FIG. 2, Table 3).Assessment of the D-D4FC susceptibility of virus from selection #2passage 20 (p20 D4FC #2) demonstrated that p20 D-D4FC#2 is 5.3-fold lesssensitive to D-D4FC than wild type as demonstrated by an increase in theEC₅₀ from 0.21 μM to 1.1 μM (FIG. 2, Table 3).

Genotyping of resistant virus. vRNA from p37 D-D4FC#1 and p20 D-D4FC#2was isolated and subjected to amplification by RT-PCR. Sequencing of thePCR product revealed the presence of three novel mutations in p37D-D4FC#I: K70N (AAA→4AAT), V901 (GTT→ATT) and RI 72K (AGA→AAA) (Table2). Two different mutations were identified in p20 D-D4FC#2: K65R(AAA→AGA) and V179D (GTT→GAT) (Table 3). No other mutations were foundin the RT genes from these viruses (from amino acids 8–330).Additionally, no mutations were found in the control viruses passaged inparallel in the absence of drug.

The isolation of D-D4FC resistant viruses with different associatedmutations could be the result of the different selection techniques usedto isolate D-D4FC resistant virus. In selection #1, the selectivepressure was removed for one cycle of infection prior to increasing thedrug concentration. This was not done during the second selectionprocedure. Additionally, the starting concentration of D-D4FC used foreach of the selection procedures varied approximately 3-fold. A muchhigher starting concentration of drug was used for selection #1 (0.75μM). These two differences are likely the cause of the differentmutations seen in the selected viruses.

Mutant recombinant HIV-1. Mutant recombinant HIV-1 containing themutations identified in the selected viruses were generated via sitedirected mutagenesis. Table 4 lists each of the mutant recombinantviruses generated as well as the corresponding EC₅₀. While thexxHIV-1_(LA1), K65R virus demonstrated a 3.9-fold decrease insusceptibility to D-D4FC, none of the other viruses showed >3.0 foldresistance. It should be noted, however, that the triple mutant(xxHIV-1_(LA1), K70N/V90I/RI72K) did not grow well, and this could bethe cause of the inability to reproduce the resistance observed in thein vitro selected virus.

TABLE 3 Susceptibility and Associated Mutations of D-D4FC Selected Virusin MT-2 Cells Mutations from Virus EC₅₀ (μM) Fold Resistance BaselineHIV-1_(LAI)p0 0.21 — — HIV-1p37 4.07 19.4 K70N D-Fd4C#1 V90I R172KHIV-1p20 1.11  5.3 K65R D-Fd4C#2 V179D

TABLE 4 D-D4FC Susceptibility of Recombinant HIV-1 in MT-2 Cells VirusEC₅₀ Fold Resistance xxLAI 0.32 — xxK65R 1.24 3.9 xxLAI 0.17 — xxK70N0.24 1.4 xxV90I 0.25 1.5 xxLAI 0.28 — xxR172K 0.23 0.8 xxV90I/R172K 0.361.3 xxLAI 0.13 — xxK70N/V90I/R172K 0.056 0.4

Table 5 provides the median effective concentration and combinationsindex (C.I.) values for D-D4FC alone and in combination with AZT and D4Tin acutely infected human PBM cells (Day6). Table 6 describes the effectof β-D and β-L-D4FC against HIV-1 and cloned viruses I human PBM cells.

TABLE 5 Median effective concentration and combinations index (C.I.)values for D-D4FC alone and in combination with AZT and D4T in acutelyinfected human PBM cells (Day 6). Treatment Parameter^(a) C.I. at F_(a)^(b) of (drug ratio) m ± SE EC₅₀ (μM) EC₉₀ (μM) r 0.50 0.75 0.90 0.95D-D4FC 0.95 ± 0.17 0.0076 0.078 0.97 AZT 0.96 ± 0.17 0.0033 0.032 0.98D4T 0.82 ± 0.16 0.015 0.22 0.95 D-D4FC/AZT 0.65 ± 0.09 0.0012 0.035 0.960.164 ± 0.134 0.276 ± 0.233 0.465 ± 0.407 0.664 ± 0.598 (100:1) 0.163 ±0.120 0.274 ± 0.215 0.460 ± 0.384 0.654 ± 0.571 D-D4FC/D4T 0.86 ± 0.110.0087 0.11 0.98 1.14 1.25 1.38 1.47 ± 1.44 (5:1) 1.045 1.154 1.276 1.37± 1.27 ^(a)m is the slope ± S.E., EC₅₀ is the median effectiveconcentration, and r is the correlation coefficient, as determined fromthe median effect plot. ^(b)C.I. <1, equal to 1 or >1 indicates synergy,additivity and antagonism. F_(a) is a componet of the median effectequation referring to the fraction of the system affected (e.g., 0.50means the C.I. at a 50% reduction of RT activity). C.I. values weredetermined for a mutually non-exclusive interaction (values in italicsare for mutually exclusive interaction, which is less vigorous).

TABLE 6 Effect of β-D- and β-L-D4FC against HIV-1 and cloned viruses inhuman PBM cells β-D-D4FC β-L-D4FC Virus EC₅₀, μM EC₉₀, μM FI₅₀ FI₉₀EC₅₀, μM EC₉₀, μM FI₅₀ FI₉₀ HIV-1_(LAV) 0.22 1.32 — — 0.034 0.16 — —xxBRU_(PITT) 0.065 0.46 — — 0.025 0.088 — — M184V_(PITT) 0.14 1.04 2 23.66 14.3 146 163 4X AZT_(PITT) 0.072 0.46 1 1 0.016 0.11 1 1 (67N, 70R,215Y, 219Q) T215Y_(PITT) 0.067 0.35 1 1 0.0047 0.03 0.2 0.3M184V/T215Y_(PITT) 0.057 0.47 1 1 8.3 74.6 332 848 215/41_(PITT) 0.130.49 2 1 0.018 0.066 1 1 4xM184V_(PITT) ^(a) 0.045 0.2 >3.3 >47 (184V,67N, 70R, 215Y, 219Q) G2-2_(PITT) ^(a) 0.04 0.2 >3.3 >47 (103N, 41L,184V, 210W, others^(b)) L-D4FC-res. (25 μM) ND ND ND ND 7.56 33.3 222208 FI₅₀ = EC₅₀ data from resistant virus EC₅₀ data from HIV-1_(LAV) orxxBRU_(PITT.) FI₉₀ = EC₉₀ data from resistant virus EC₉₀ data fromHIV-1_(LAV) or xxBRU_(PITT.) Values in bold indicate FI > 10. ^(a)InMT-2 cells: Cortesy of Dr. J. Mellors (Pittsburgh) ^(b)Additionalmutations: 203D, 207A, 211K, 214F, 245M, 277N, 283I, 284K, 200D, 311RIII. Combination or Alternation HIV-Agents

In general, during alternation therapy, an effective dosage of eachagent is administered serially, whereas in combination therapy, aneffective dosage of two or more agents are administered together. Inalternation therapy, for example, one or more first agents can beadministered in an effective amount for an effective time period totreat the viral infection, and then one or more second agentssubstituted for those first agents in the therapy routine and likewisegiven in an effective amount for an effective time period.

The dosages will depend on such factors as absorption, biodistribution,metabolism and excretion rates for each drug as well as other factorsknown to those of skill in the art. It is to be noted that dosage valueswill also vary with the severity of the condition to be alleviated. Itis to be further understood that for any particular subject, specificdosage regimens and schedules should be adjusted over time according tothe individual need and the professional judgment of the personadministering or supervising the administration of the compositions.Examples of suitable dosage ranges for anti-HIV compounds, includingnucleoside derivatives (e.g. AZT, D4T, DDI, and 3TC) or proteaseinhibitors, for example, nelfinavir and indinavir, can be found in thescientific literature and in the Physicians Desk Reference. Manyexamples of suitable dosage ranges for other compounds described hereinare also found in public literature or can be identified using knownprocedures. These dosage ranges can be modified as desired to achieve adesired result.

In one preferred embodiment, D-D4FC is administered in combination witha protease inhibitor. In particular embodiments, D-D4FC is administeredin combination or alternation with indinavir (Crixivan), nelfinavir([3S-[2(2S*,3S*),3-alpha,4-a-beta,8a-beta-]]-N-(1,1-dimethylethyl)decahydro-2-)2-hydrozy-3-[(3-hydrozy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolincarboxamidemono-methanesulfonate) (Viracept), saquinavir (Invirase), or 141W94(amprenavir;(S)-tetrahydrofuran-3-yl-N-[(1S,2R)-3-[N-[(4-aminophenyl)sulfonyl]-N-isobutylamino]-1-benzyl-2-hydroxypropyl]carbamate;or(S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one(efavirenz).

In another preferred embodiment, D-D4FC is administered in combinationor alternation with a nucleoside analog, including abacavir (1592U89)which is(1S,4R)-4-[2-amino-6-cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsuccinate.

In another embodiment, D-D4FC is administered in combination with anonnucleoside reverse transcriptase inhibitor such as DMP-266((S)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one(SUSTIVA, see U.S. Pat. No. 5,519,021); delavirdine,(1-[3-(1-methyl-ethyl)amino]-2-pyridinyl-4-[[5-[(methylsulfonyl)amino]-1H-indol-2-yl]carbonyl]-,monoethanesulfonate),nevirapine, or delarvirdine.

In other embodiments, D-D4FC is administered in combination oralternation with an HIV-integrase inhibitor or a chemokine inhibitor.

II. Analysis of D-D4FC Induced Mutation of HIV Genome

Methods and kits similar to those described in U.S. Pat. No. 5,409,810to Larder et al, for AZT, but based on the mutation profile for D-D4FC,can be used to analyze for the presence of D-D4FC induced mutations andthus form part of the invention presented herein. In addition toincorporating the Larder patent by reference in its entirety, thesetechniques are set out below.

In one aspect of the invention there is provided a method of assessingthe sensitivity of an HIV-1 sample to D-D4FC, which includes:

-   -   (i) isolating nucleic acid from the sample,    -   (ii) hybridizing an oligonucleotide to the nucleic acid, the        oligonucleotide being complementary to a region of the wild-type        DNA sequence (or its corresponding RNA) or to a region of the        mutant DNA sequence (or its corresponding RNA);    -   (iii) attempting polymerization of the nucleic acid from the        3′-end of the oligonucleotide,    -   (iv) ascertaining whether or not an oligonucleotide primer        extended product is present.

It is possible to use genomic DNA or RNA isolated from HIV-1 samples inthis methodology. Suitable cells for supporting the growth of HIV-1isolate are incubated for a period of time. The cells are recovered bycentrifugation. DNA can then be isolated by digestion of the cells withproteinase K in the presence of EDTA and a detergent such as SDS,followed by extraction with phenol.

Well-known extraction and purification procedures are available for theisolation of DNA from a sample. RNA can be isolated using the followingmethodology. Suitable cells are being infected and incubated for aperiod of time. The cells are recovered by centrifugation. The cells areresuspended in an RNA extraction buffer followed by digestion using aproteinase digestion buffer and digestion with proteinase K. Proteinsare removed in the presence of a phenol/chloroform mixture. RNA can thenbe recovered following further centrifugation steps. (Maniatis, T., etal, Molecular Coning, A laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, (1989)).

Although it is possible to use unamplified nucleic acid, due to therelative scarcity of nucleic acid in an HIV-1 sample it is preferable toamplify it. Nucleic acid may be selectively amplified using thetechnique of polymerase chain reaction (PCR), which is an in vitromethod for producing large amounts of specific nucleic acid fragment ofdefined length and sequence from small amounts of a template.

The PCR is comprised of standard reactants using Mg²+ concentration,oligonucleotide primers and temperature cycling conditions foramplification of the RT gene using the primers. The primers are chosensuch that they will amplify the entire RT gene or a selected sequencewhich incorporates nucleotides corresponding to a region of thewild-type DNA sequence of HIV-1 that includes the codon which ismutated.

RNA cannot be amplified directly by PCR. Its corresponding cDNA must besynthesized. Synthesis of cDNA is normally carried out by primed reversetranscription using oligo-dT primers. Advantageously, primers are chosensuch that they will simplify the nucleic acid sequence for RT or aselected sequence which incorporates nucleotides corresponding to theregion of RNA corresponding to the wild-type DNA sequence or to theregion of the mutant DNA sequence corresponding to the 70th (K to N),90th or 172th codon of the reverse transcriptase region. This could beachieved by preparing an oligonucleotide primer which is complementaryto a region of the RNA strand which is upstream of the correspondingsequence of the wild-type DNA sequence. cDNA prepared by thismethodology (see Maniatis, T., et al., supra.) can then be used in thesame way as for the DNA already discussed.

The next stage of the methodology is to hybridize to the nucleic acid anoligonucleotide which is complementary to a region of the wild-type DNAsequence (or its corresponding RNA) or to a region of the mutant DNAsequence (or its corresponding RNA).

Conditions and reagents are then provided to permit polymerization ofthe nucleic acid from the 3′-end of the oligonucleotide primer. Suchpolymerization reactions are well-known in the art.

If the oligonucleotide primer has at its 3′-end a nucleotide which iscomplementary to a mutant genotype, that is a genotype which has anucleotide change at the 70th (K to N), 90th or 172th codon in the RTregion, then polymerization of the nucleic acid sequence will only occurif the nucleic acid of the sample is the same as the mutant genotype.Polymerization of a wild type nucleic acid sequence will not occur or atleast not to a significant extent because of a mis-match of nucleotidesat the 3′-end of the oligonucleotide primer and the nucleic acidsequence of the sample.

If the oligonucleotide primer has at its 3′-end of nucleotide which iscomplementary to the wild-type genotype, that is a genotype which hasthe wild-type nucleotide at the 70th (K to N), 90th or 172th codon inthe RT region, then there will be polymerization of a nucleic acidsequence which is wild-type at that position. There will be nopolymerization of a nucleic acid which has a mutant nucleotide at the3′-position.

The preferred length of each oligonucleotide is 15–20 nucleotides. Theoligonucleotide can be prepared according to methodology well known tothe man skilled in the art (Koster, H., Drug Research, 30 p548 (1980);Koster, H., Tetrahedron Letters p1527 (1972); Caruthers, TetrahedronLetters, p719, (1980); Tetrahedron Letters, p1859, (1981); TetrahedronLetters 24, p245, (1983); Gate. M. Nucleic Acid Research, 8, p1081,(1980)) and is generally prepared using an automated DNA synthesizersuch as an Applied Biosystems 381A synthesizer.

It is convenient to determine the presence of an oligonucleotide primerextended product. The means for carrying out the detection is by usingan appropriate label.

The label may be conveniently attached to the oligonucleotide primer orto some other molecule which will bind the primer extendedpolymerization product.

The label may be for example an enzyme, radioisotope or fluorochrome. Apreferred label may be biotin which could be subsequently detected usingstreptavidin conjugated to an enzyme such as peroxidase or alkalinephosphatase. The presence of an oligonucleotide primer extendedpolymerization product can be detected for example by running thepolymerization reaction on an agarose gel and observing a specific DNAfragment labeled with ethidium bromide, or Southern blotted andautoradiographed to detect the presence or absence of bandscorresponding to polymerised product. If a predominant band is presentwhich corresponds only to the labeled oligonucleotide then thisindicates that polymerization has been occurred. If bands are present ofthe correct predicted size, this would indicate that polymerization hasoccurred.

For example, DNA isolated from patients' lymphocytes as described hereinis used as a template for PCR amplification using syntheticoligonucleotide primers which either match or mis-match with theamplified sequences. The feasibility of PCR in detecting such mutationshas already been demonstrated. PCR using the Amplification RefractoryMutation system (“ARMS”) (Newton, C. R., et al. Nucleic Acids Research,17, p2503, (1989)) Synthetic oligonucleotide are produced that anneal tothe regions adjacent to an including the specific mutations such thatthe 3′ENDE of the oligonucleotide either matches of mismatches with amutant or wild-type sequence. PCR is carried out which results in theidentification of a DNA fragment (using gel electrophoresis) where amatch has occurred or no fragment where a mismatch occurred.

DNA is extracted from HIV-1 infected T-cells as described herein andsubjected to “ARMS” PCR analysis using these primers.

The presence of a fragment is identified by using an oligonucleotideprimer as described above, i.e., by attempting polymerisation using anoligonucleotide primer which may be labeled for the amplified DNAfragment under stringent conditions which only allow hybridization ofcomplementary DNA (the only difference is that differentialhybridization does not have to be performed as fragments of DNAamplified by the “ARMS” method will be the same whether derived frommutant or wild-type DNS, so a common oligonucleotide can be used todetect the presence of these fragments. The sequence of such anoligonucleotide is derived from a DNA sequence within the DNA fragmentthat is conserved amongst HIV-1 strains).

The above PCR assay may be adapted to enable direct detection ofmutations associated with D-D4FC resistance in DNA from PBL samples frominfected individuals that have not been cultured to obtain virus. Asthis material generally contains considerably less HIV-1 DNA than thatin infected lymphoid cultures a “double PCR” (or nested set) protocolcan be used (Simmonds, P., Balfe, P, Peutherer, J. F., Ludlam, C. A.,Bishop, J. O. and Leigh Brown, A. J., J. Virol., 64, 864–872, (1990)) toboost the amount of target HIV-1 RT DNA signal in the samples. Thedouble PCR overcomes the problem of limited amplification of a raretemplate sequence. A small amount of the pre-amplified material may beused in the second PCR with primer pairs designed to allowdiscrimination of wild type and mutant residues.

A suitable test kit for use in an assay to determine the resistancestatus of an HIV-1 sample to D-D4FC which makes use of a methodologyaccording to the first aspect of the invention, comprises anoligonucleotide being complementary to a region of the wild-type DNAsequence (or its corresponding RNA) or to a region of the mutant DNAsequence as described herein, other materials required forpolymerisation of the nucleic acid from the 3′-end of theoligonucleotide and means for determining the presence of anoligonucleotide primer extended product. Such other materials includeappropriate enzymes, buffers and washing solutions, and a label and asubstrate for the label if necessary. If PCR is used to amplify nucleicacid then additional materials such as appropriate oligonucleotideprimers which will amplify a region of the wild-type DNA sequence (orits corresponding RNA) or a region of the mutant DNA sequence asdescribed herein (or its corresponding RNA) and dNTP's should beincluded.

In a second aspect of the invention there is provided a method ofdetermining the sensitivity of an HIV-1 sample to D-D4FC whichcomprises:

-   -   (i) isolating the nucleic acid from the sample;    -   (ii) hybridizing the nucleic acid with an oligonucleotide being        complementary to a region of the wild-type DNA sequence (or its        corresponding RNA) or to a region of the mutant DNA sequence set        forth in FIG. 1 (or its corresponding RNA) containing one or        more of the nucleotides at the region of the 70th (K to N), 90th        or 1 72th codon in the RT region; and    -   (iii) ascertaining whether or not any of the resulting hybrids        of the oligonucleotide and nucleic acid have complementary        nucleotides at one of these positions.

Preferably the oligonucleotide is so designed to form a perfectlymatched hybrid with its complement.

Nucleic acid (DNA or RNA) is isolated from a sample by theaforementioned methods as described for the first aspect of theinvention.

Similarly, PCR may be used to amplify the RT DNA (or its correspondingRNA) or preferably to amplify a region of the RT DNA (or itscorresponding RNA) which incorporates DNA (or its corresponding RNA)containing one or more of the nucleotides at the designated position.

In the second stage of this methodology the nucleic acid is then used tohybridize to oligonucleotides complementary to a region of the wild-typeDNA sequence (or its corresponding (RNA) or to a region of the mutantDNA sequence.

The oligonucleotide may be of any length depending on the number ofnucleotide positions of interest which are being examined. If theoligonucleotide is designed to include a nucleotide at only one positionof interest then this nucleotide is preferably at or close to the centerposition of the oligonucleotide.

In order to ascertain whether or not the oligonucleotide and nucleicacid sequence have formed a matched hybrid, specific hybridizationconditions are set so that a hybrid is only formed when the nucleotideor nucleotides at the 70th (K to N), 90th or 172th codon of the reversetranscriptase region are complementary to the corresponding nucleotideor nucleotides of the oligonucleotide which either permits hybridizationor no hybridization. It is important to establish for example thetemperature of the reaction and the concentration of salt solutionbefore carrying out the hybridization step to find conditions that arestringent enough to guarantee specificity (Maniatis, T., et al.,Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring HarbourLaboratory Press, (1989). If the oligonucleotide probe has a DNAsequence which is complementary to a wild-type nucleic acid sequence atone or more of its nucleotides corresponding to the 70th (K to N), 90thor 172th codon in the reverse transcriptase region then thisoligonucleotide will hybridize perfectly to wild-type nucleic acid. Ifthere is no hybridize perfectly to wild-type nucleic acid. If there isno hybridization then this would suggest that the nucleic acid isolatedfrom the same contains one or more mutations.

If the oligonucleotide probe has a DNA sequence which is complementaryto a mutant nucleic acid sequence then this oligonucleotide willhybridize to mutant nucleic acid. If there is no hybridization thiswould suggest that the nucleic acid isolated from the sample contains nosuch mutation or mutations. The oligonucleotide probes may be labeled asa means of detection as for the first aspect of the invention.

The hybridization and subsequent removal of non-hybridized nucleic acidsare performed under stringent conditions which only allow hybridizationof the complementary DNA and not the oligonucleotide containing amismatch (i.e. oligonucleotide specific hybridization as described forthe detection of sickle cell mutation using the β-globin or HLA-DQα gene(Saikt, R. K., et al., Nature, 324, p163, (1986), the activated Ras gene(Ver Laan-de, Vries, M., et al., Gene, 50, 313, (1986)) andβ-thalassaemia Wong, C., et al., Nature, 330, p384, (1987)).

The hybridization may be carried out by immobilization of the RT nucleicacid sequence onto nitrocellulose, nylon or other solid matrix (e.g.dot-blot). It is convenient to determine the presence of an hybrid byusing the means of a label. For example, the chemically synthesizedoligonucleotide probes can be suitably labeled using enzyme,radioisotope or fluorochrome. A preferred label may be biotin whichcould be subsequently detected using streptavidin conjugated to anenzyme such as peroxidase or alkaline phosphatase.

Alternatively the hybridization may be carried out by immobilization ofthe chemically synthesized oligonucleotides referred to above, which areunlabeled, onto a solid support referred to above and subsequenthybridization by a labeled RT nucleic acid sequence as describedpreviously.

In both situations described above for hybridization suitable controlreactions will be incorporated to determine that efficient hybridizationhas occurred. (e.g., the hybridization of oligonucleotides to acomplementary oligonucleotide).

Results would be readily interpreted as the isolated nucleic acid wouldhybridize to either the wild type oligonucleotide or the mutantoligonucleotide.

A suitable test kit for use in an assay to determine the sensitivity ofan HIV-1 sample to D-D4FC which makes use of a methodology according tothe second aspect of the invention comprises an oligonucleotide beingcomplementary to a region of the wild-type DNA sequence (or itscorresponding RNA) or to the pertinent region of the mutant DNAsequence, along with other materials required to permit hybridization.Such materials include appropriate buffers and washing solutions and alabel and a substrate for the label if necessary. Normally theoligonucleotide would be labeled. If PCR is used to amplify nucleic acidprior to hybridization then additional materials such as appropriateoligonucleotide primers which will amplify a region of the wild-type DNAsequence (or its corresponding RNA) or a region of the mutant DNAsequence, appropriate enzymes and dNTP's (deoxy nucleotidetriphosphates) should be included.

In one alternate format of the assay, the dNTP's in the amplificationmay or may not be coupled to a detector molecule such as a radioisotope,biotin, fluorochrome or enzyme.

It is also possible to detect zidovudine resistant mutations in theHIV-1 RT RNA isolated from clinical samples using an RNA amplificationsystem. Using the methodology described by Guatelli et al. (Proc. Natl.Acad. Sci, (USA), 8/7, 1874–1878, (March 1990)) a target nucleic acidsequence can be replicated (amplified) exponentially in vitro underisothermal conditions by using three enzymatic activities essential toretroviral replication: reverse transcriptase, RNase H and aDNA-dependant RNA polymerase. Such a methodology may be employedfollowed by an hybridization step to distinguish mutant from wild-typenucleotides at discussed previously.

Preparation of Pharmaceutical Compositions

Humans suffering from effects caused by any of the diseases describedherein, and in particular, HIV infection, can be treated byadministering to the patient an effective amount of D-D4FC or apharmaceutically acceptable salt or prodrug thereof in the presence of apharmaceutically acceptable carrier or diluent, for any of theindications or modes of administration as described in detail herein.The active materials can be administered by any appropriate route, forexample, orally, parenterally, enterally, intravenously, intradermally,subcutaneously, transdermally, intranasally or topically, in liquid orsolid form.

The active compound(s) are included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount of compound to inhibit viralreplication in vivo, especially HIV replication, without causing serioustoxic effects in the treated patient. By “inhibitory amount” is meant anamount of active ingredient sufficient to exert an inhibitory effect asmeasured by, for example, an assay such as the ones described herein.

A preferred dose of the compound for all the above-mentioned conditionswill be in the range from about 1 to 75 mg/kg, preferably 1 to 20 mg/kg,of body weight per day, more generally 0.1 to about 100 mg per kilogrambody weight of the recipient per day. The effective dosage range of thepharmaceutically acceptable derivatives can be calculated based on theweight of the parent nucleoside to be delivered. If the derivativeexhibits activity in itself, the effective dosage can be estimated asabove using the weight of the derivative, or by other means known tothose skilled in the art.

The compounds are conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50 to 1000 mg is usually convenient.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.02 to 70micromolar, preferably about 0.5 to 10 mM. This may be achieved, forexample, by the intravenous injection of a 0.1 to 25% solution of theactive ingredient, optionally in saline, or administered as a bolus ofthe active ingredient.

The concentration of active compound in the drug composition will dependon absorption, distribution, metabolism and excretion rates of the drugas well as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bind agents,and/or adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compounds can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compounds or their pharmaceutically acceptable derivative or saltsthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, proteaseinhibitors, or other nucleoside or non-nucleoside antiviral agents, asdiscussed in more detail above. Solutions or suspensions used forparental, intradermal, subcutaneous, or topical application can includethe following components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The parentalpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers, these may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Patent No. 4,522,811. For example, liposome formulations may beprepared by dissolving appropriate lipid(s) (such as stearoylphosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoylphosphatidyl choline, and cholesterol) in an inorganic solvent that isthen evaporated, leaving behind a thin film of dried lipid on thesurface of the container. An aqueous solution of the active compound orits monophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

Controlled Release Formulations

All of the U.S. patents cited in this section on controlled releaseformulations are incorporated by reference in their entirety.

The field of biodegradable polymers has developed rapidly since thesynthesis and biodegradability of polylactic acid was reported byKulkarni et al., in 1966 (“Polylactic acid for surgical implants,” Arch.Surg., 93:839). Examples of other polymers which have been reported asuseful as a matrix material for delivery devices include polyanhydrides,polyesters such as polyglycolides and polylactide-co-glycolides,polyamino acids such as polylysine, polymers and copolymers ofpolyethylene oxide, acrylic terminated polyethylene oxide, polyamides,polyurethanes, polyorthoesters, polyacrylonitriles, andpolyphosphazenes. See, for example, U.S. Pat. Nos. 4,891,225 and4,906,474 to Langer (polyanhydrides), U.S. Pat. No. 4,767,628 toHutchinson (polylactide, polylactide-co-glycolide acid), and U.S. Pat.No. 4,530,840 to Tice, et al. (polylactide, polyglycolide, andcopolymers). See also U.S. Pat. No. 5,626,863 to Hubbell, et al whichdescribes photopolymerizable biodegradable hydrogels as tissuecontacting materials and controlled release carriers (hydrogels ofpolymerized and crosslinked macromers comprising hydrophilic oligomershaving biodegradable monomeric or oligomeric extensions, which are endcapped monomers or oligomers capable of polymerization andcrosslinking); and PCT WO 97/05185 filed by Focal, Inc. directed tomultiblock biodegradable hydrogels for use as controlled release agentsfor drug delivery and tissue treatment agents.

Degradable materials of biological origin are well known, for example,crosslinked gelatin. Hyaluronic acid has been crosslinked and used as adegradable swelling polymer for biomedical applications (U.S. Pat. No.4,957,744 to Della Valle et. al.; (1991) “Surface modification ofpolymeric biomaterials for reduced thrombogenicity,” Polym. Mater. Sci.Eng., 62:731–735]).

Many dispersion systems are currently in use as, or being explored foruse as, carriers of substances, particularly biologically activecompounds. Dispersion systems used for pharmaceutical and cosmeticformulations can be categorized as either suspensions or emulsions.Suspensions are defined as solid particles ranging in size from a fewmanometers up to hundreds of microns, dispersed in a liquid medium usingsuspending agents. Solid particles include microspheres, microcapsules,and nanospheres. Emulsions are defined as dispersions of one liquid inanother, stabilized by an interfacial film of emulsifiers such assurfactants and lipids. Emulsion formulations include water in oil andoil in water emulsions, multiple emulsions, microemulsions,microdroplets, and liposomes. Microdroplets are unilamellar phospholipidvesicles that consist of a spherical lipid layer with an oil phaseinside, as defined in U.S. Pat. Nos. 4,622,219 and 4,725,442 issued toHaynes. Liposomes are phospholipid vesicles prepared by mixingwater-insoluble polar lipids with an aqueous solution. The unfavorableentropy caused by mixing the insoluble lipid in the water produces ahighly ordered assembly of concentric closed membranes of phospholipidwith entrapped aqueous solution.

U.S. Pat. No. 4,938,763 to Dunn, et al., discloses a method for formingan implant in situ by dissolving a nonreactive, water insolublethermoplastic polymer in a biocompatible, water soluble solvent to forma liquid, placing the liquid within the body, and allowing the solventto dissipate to produce a solid implant. The polymer solution can beplaced in the body via syringe. The implant can assume the shape of itssurrounding cavity. In an alternative embodiment, the implant is formedfrom reactive, liquid oligomeric polymers which contain no solvent andwhich cure in place to form solids, usually with the addition of acuring catalyst.

A number of patents disclose drug delivery systems that can be used toadminister D-D4FC or a nucleotide or other defined prodrug thereof. U.S.Pat. No. 5,749,847 discloses a method for the delivery of nucleotidesinto organisms by electrophoration. U.S. Pat. No. 5,718,921 disclosesmicrospheres comprising polymer and drug dispersed there within. U.S.Pat. No. 5,629,009 discloses a delivery system for the controlledrelease of bioactive factors. U.S. Pat. No, 5,578,325 disclosesnanoparticles and microparticles of non-linear hydrophilic hydrophobicmultiblock copolymers. U.S. Pat. No. 5,545,409 discloses a deliverysystem for the controlled release of bioactive factors. U.S. Pat. No.5,494,682 discloses ionically cross-linked polymeric microcapsules.

U.S. Pat. No. 5,728,402 to Andrx Pharmaceuticals, Inc. describes acontrolled release formulation that includes an internal phase whichcomprises the active drug, its salt or prodrug, in admixture with ahydrogel forming agent, and an external phase which comprises a coatingwhich resists dissolution in the stomach. U.S. Pat. Nos. 5,736,159 and5,558,879 to Andrx Pharmaceuticals, Inc. discloses a controlled releaseformulation for drugs with little water solubility in which a passagewayis formed in situ. U.S. Pat. No. 5,567,441 to Andrx Pharmaceuticals,Inc. discloses a once-a-day controlled release formulation. U.S. Pat.No. 5,508,040 discloses a multiparticulate pulsatile drug deliverysystem. U.S. Pat. No. 5,472,708 discloses a pulsatile particle baseddrug delivery system. U.S. Pat. No. 5,458,888 describes a controlledrelease tablet formulation which can be made using a blend having aninternal drug containing phase and an external phase which comprises apolyethylene glycol polymer which has a weight average molecular weightof from 3,000 to 10,000. U.S. Pat. No. 5,419,917 discloses methods forthe modification of the rate of release of a drug form a hydrogel whichis based on the use of an effective amount of a pharmaceuticallyacceptable ionizable compound that is capable of providing asubstantially zero-order release rate of drug from the hydrogel. U.S.Pat. No. 5,458,888 discloses a controlled release tablet formulation.

U.S. Pat. No. 5,641,745 to Elan Corporation, plc discloses a controlledrelease pharmaceutical formulation which comprises the active drug in abiodegradable polymer to form microspheres or nanospheres. Thebiodegradable polymer is suitably poly-D,L-lactide or a blend ofpoly-D,L-lactide and poly-D,L-lactide-co-glycolide. U.S. Pat. No.5,616,345 to Elan Corporation plc describes a controlled absorptionformulation for once a day administration that includes the activecompound in association with an organic acid, and a multi-layer membranesurrounding the core and containing a major proportion of apharmaceutically acceptable film-forming, water insoluble syntheticpolymer and a minor proportion of a pharmaceutically acceptablefilm-forming water soluble synthetic polymer. U.S. Pat. No. 5,641,515discloses a controlled release formulation based on biodegradablenanoparticles. U.S. Pat. No. 5,637,320 discloses a controlled absorptionformulation for once a day administration. U.S. Pat. Nos. 5,580,580 and5,540,938 are directed to formulations and their use in the treatment ofneurological diseases. U.S. Pat. No. 5,533,995 is directed to a passivetransdermal device with controlled drug delivery. U.S. Pat. No.5,505,962 describes a controlled release pharmaceutical formulation.

Prodrug Formulations

D-D4FC or any of the nucleosides or other compounds which are describedherein for use in combination or alternation therapy with D-D4FC or itsrelated compounds can be administered as an acylated prodrug or anucleotide prodrug, as described in detail below.

Any of the nucleosides described herein or other compounds that containa hydroxyl or amine function can be administered as a nucleotide prodrugto increase the activity, bioavailability, stability or otherwise alterthe properties of the nucleoside. A number of nucleotide prodrug ligandsare known. In general, alkylation, acylation or other lipophilicmodification of the hydroxyl group of the compound or of the mono, di ortriphosphate of the nucleoside will increase the stability of thenucleotide. Examples of substituent groups that can replace one or morehydrogens on the phosphate moiety or hydroxyl are alkyl, aryl, steroids,carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Manyare described in R. Jones and N. Bischofberger, Antiviral Research, 27(1995) 1–17. Any of these can be used in combination with the disclosednucleosides or other compounds to achieve a desire effect.

The active nucleoside or other hydroxyl containing compound can also beprovided as an ether lipid (and particularly a 5′-ether lipid for anucleoside), as disclosed in the following references, Kucera, L. S., N.Iyer, E. Leake, A. Raben, Modest E. K., D. L. W., and C. Piantadosi.1990. “Novel membrane-interactive ether lipid analogs that inhibitinfectious HIV-1 production and induce defective virus formation.” AIDSRes. Hum. Retro Viruses. 6:491–501; Piantadosi, C., J. Marasco C. J., S.L. Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L.S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV activity.” J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.Richrnan, D. A. Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. vanden Bosch. 1992. “Greatly enhanced inhibition of human immunodeficiencyvirus type 1 replication in CEM and HT4-6C cells by 3′-deoxythymidinediphosphate dimyristoylglycerol, a lipid prodrug of 3,-deoxythymidine.”Antimicrob. Agents Chemother. 36:2025.2029; Hostetler, K. Y., L. M.Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman, 1990.“Synthesis and antiretroviral activity of phospholipid analogs ofazidothymidine and other antiviral nucleosides.” J. Biol. Chem.265:61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside orother hydroxyl or amine containing compound, preferably at the 5′-OHposition of the nucleoside or lipophilic preparations, include U.S. Pat.No. 5,149,794 (Sep. 22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654(Mar. 16, 1993, Hostetler et al., U.S. Pat. No. 5,223,263 (Jun. 29,1993, Hostetler et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvinet al.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvinet al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996; Basava et-al.),Foreign patent applications that disclose lipophilic substituents thatcan be attached to the nucleosides of the present invention, orlipophilic preparations, include WO 89/02733, WO 90/00555, WO 91/16920,WO 91/18914, WO 93/00910, WO 94/26273, WO 96/15132, EP 0 350 287, EP93917054.4, and WO 91/19721.

Nonlimiting examples of nucleotide prodrugs are described in thefollowing references: Ho, D. H. W. (1973) “Distribution of Kinase anddeaminase of 1β-D-arabinofuranosylcytosine in tissues of man and muse.”Cancer Res. 33, 2816–2820; Holy, A. (1993) Isopolar phosphorous-modifiednucleotide analogues,” In: De Clercq (Ed.), Advances in Antiviral DrugDesign, Vol. I, JAI Press, pp. 179–231; Hong, C. I., Nechaev, A., andWest, C. R. (1979a) “Synthesis and antitumor activity of1β-D-arabino-furanosylcytosine conjugates of cortisol and cortisone.”Biochem. Biophys. Rs. Commun. 88, 1223–1229; Hong, C. I., Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) “Nucleosideconjugates as potential antitumor agents. 3. Synthesis and antitumoractivity of 1-(β-D-arabinofuranosyl)cytosine conjugates ofcorticosteroids and selected lipophilic alcohols.” J. Med. Chem. 28,171–177; Hosteller, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van denBosch, H. and Richman J Biol. Chem. 265, 6112–6117; Hosteller, K. Y.,Carson, D. A. and Richman, D. D. (1991); “Phosphatidylazidothymidine:mechanism of antiretroviral action in CEM cells.” J. Biol Chem. 266,11714–11717; Hosteller, K. Y., Korba, B. Sridhar, C., Gardener, M.(1994a) “Antiviral activity of phosphatidyl-dideoxycytidine in hepatitisB-infected cells and enhanced hepatic uptake in mice.” Antiviral Res.24, 59–67; Hosteller, K. Y., Richman, D. D., Sridhar. C. N. Felgner, P.L. Felgner, J., Ricci, J., Gardener, M. F. Selleseth, D. W. and Ellis,M. N. (1994b) “Phosphatidylazidothymidine and phosphatidyl-ddC:Assessment of uptake in mouse lymphoid tissues and antiviral activitiesin human immunodeficiency virus-infected cells and in rauscher leukemiavirus-infected mice.” Antimicrobial Agents Chemother. 38, 2792–2797;Hunston, R. N., Jones, A. A. McGuigan, C., Walker, R. T., Balzarini, J.,and DeClercq, E. (1984) “Synthesis and biological properties of somecyclic phosphotriesters derived from 2′-deoxy-5-fluorouridine.” J. Med.Chem. 27,440–444; Ji, Y. H., Moog, C., Schmitt, G., Bischoff, P. andLuu, B. (1990); “Monophosphoric acid esters of 7-β-hydroxycholesteroland of pyrimidine nucleoside as potential antitumor agents: synthesisand preliminary evaluation of antitumor activity.” J. Med. Chem. 332264–2270; Jones, A. S., McGuigan, C., Walker, R. T., Balzarini, J. andDeClercq, E. (1984) “Synthesis, properties, and biological activity ofsome nucleoside cyclic phosphoramidates.” J. Chem. Soc. Perkin Trans. I,1471–1474; Juodka, B. A. and Smart, J. (1974) “Synthesis ofdiribonucleoside phosph (P→N) amino acid derivatives.” Coll. Czech.Chem. Comm. 39, 363–968; Kataoka, S., Imai, J., Yamaji, N., Kato, M.,Saito, M., Kawada, T. and Imai, S. (1989) “Alkylated cAMP derivatives;selective synthesis and biological activities.” Nucleic Acids Res. Sym.Ser. 21, 1–2; Kataoka, S., Uchida, “(cAMP) benzyl and methyl triesters.”Heterocycles 32, 1351–1356; Kinchington, D., Harvey, J. J., O'Connor, T.J., Jones, B. C. N. M., Devine, K. G., Taylor-Robinson D., Jeffries, D.J. and McGuigan, C. (1992) “Comparison of antiviral effects ofzidovudine phosphoramidate and phosphorodiamidate derivatives againstHIV and ULV in vitro.” Antiviral Chem. Chemother. 3, 107–112; Kodama,K., Morozumi, M., Saithoh, K. I., Kuninaka, H., Yosino, H. andSaneyoshi, M. (1989) “Antitumor activity and pharmacology of1-β-D-arabinofuranosylcytosine -5′-stearylphosphate; an orally activederivative of 1-β-Darabinofuranosylcytosine.” Jpn. J. Cancer Res. 80,679–685; Korty, M. and Engels, J. (1979) “The effects of adenosine- andguanosine 3′,5′ phosphoric and acid benzyl esters on guinea-pigventricular myocardium.” Naunyn-Schmiedeberg's Arch. Pharmacol. 310,103–111; Kumar, A., Goe, P. L., Jones, A. S. Walker, R. T. Balzarini, J.and DeClercq, E. (1990) “Synthesis and biological evaluation of somecyclic phosphoramidate nucleoside derivatives.” J. Med. Chem, 33,2368–2375; LeBec, C., and Huynh-Dinh, T. (1991) “Synthesis of lipophilicphosphate triester derivatives of 5-fluorouridine an arabinocytidine asanticancer prodrugs.” Tetrahedron Lett. 32, 6553–6556; Lichtenstein, J.,Barner, H. D. and Cohen, S. S. (1960) “The metabolism of exogenouslysupplied nucleotides by Escherichia coli.,” J. Biol. Chem. 235, 457–465;Lucthy, J., Von Daeniken, A., Friederich, J. Manthey, B., Zweifel, J.,Schlatter, C. and Benn, M. H. (1981) “Synthesis and toxicologicalproperties of three naturally occurring cyanoepithioalkanes”. Mitt. Geg.Lebensmittelunters. Hyg. 72, 131–133 (Chem. Abstr. 95, 127093); McGigan,C. Tollerfield, S. M. and Riley, P. a. (1989) “Synthesis and biologicalevaluation of some phosphate triester derivatives of the anti-viral drugAra.” Nucleic Acids Res. 17, 6065–6075; McGuigan, C., Devine, K. G.,O'Connor, T. J., Galpin, S. A., Jeffries, D. J. and Kinchington, D.(1990a) “Synthesis and evaluation of some novel phosphoramidatederivatives of 3′-azido-3′-deoxythymidine (AZT) as anti-HIV compounds.”Antiviral Chem. Chemother. 1 107–113; McGuigan, C., O'Connor, T. J.,Nicholls, S. R. Nickson, C. and Kinchington, D. (1990b) “Synthesis andanti-HIV activity of some novel substituted dialkyl phosphatederivatives of AZT and ddCyd.” Antiviral Chem. Chemother. 1, 355–360;McGuigan, C., Nicholls, S. R., O'Connor, T. J., and Kinchington, D.(1990c) “Synthesis of some novel dialkyl phosphate derivative of3′-modified nucleosides as potential anti-AIDS drugs.” Antiviral Chem.Chemother. 1, 25–33; McGuigan, C., Devin, K. G., O'Connor, T. J., andKinchington, D. (1991) “Synthesis and anti-HIV activity of somehaloalkyl phosphoramidate derivatives of 3′-azido-3′deoxythylmidine(AZT); potent activity of the trichloroethyl methoxyalaninyl compound.”Antiviral Res. 15, 255–263; McGuigan, C., Pathirana, R. N., Balzarini,J. and DeClercq, E. (1993b) “Intracellular delivery of bioactive AZTnucleotides by aryl phosphate derivatives of AZT.” J. Med. Chem. 36,1048–1052.

Alkyl hydrogen phosphate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5, 271–277; Meyer, R. B., Jr., Shuman, D. A. and Robins, R.K. (1973) “Synthesis of purine nucleoside 3′,5′-cyclicphosphoramidates.” Tetrahedron Lett. 269–272; Nagyvary, J. Gohil, R. N.,Kirchner, C. R. and Stevens, J. D. (1973) “Studies on neutral esters ofcyclic AMP,” Biochem. Biophys. Res. Commun. 55, 1072–1077; Namane, A.Gouyette, C., Fillion, M. P., Fillion, G. and Huynh-Dinh, T. (1992)“Improved brain delivery of AZT using a glycosyl phosphotriesterprodrug.” J. Med. Chem. 35, 3039–3044; Nargeot, J. Nerbonne, J. M.Engels, J. and Leser, H. A. (1983) Natl. Acad. Sci. U.S.A. 80,2395–2399; Nelson, K. A., Bentrude, W. G. Stser, W. N. and Hutchinson,J. P. (1987) “The question of chair-twist equilibria for the phosphaterings of nucleoside cyclic 3′,5′monophosphates. ¹HNMR and x-raycrystallographic study of the diastereomers of thymidine phenyl cyclic3′,5′-monophosphate.” J. Am. Chem. Soc. 109, 4058–4064; Nerbonne, J. M.,Richard, S., Nargeot, J. and Lester, H. A. (1984) “New photoactivatablecyclic nucleotides produce intracellular jumps in cyclic AMP and cyclicGMP concentrations.” Nature 301, 74–76; Neumann, J. M., Herv_, M.,Debouzy, J. C., Guerra, F. I., Gouyette, C., Dupraz, B. and Huyny-Dinh,T. (1989) “Synthesis and transmembrane transport studies by NMR of aglucosyl phospholipid of thymidine.” J. Am. Chem. Soc. 111, 4270–4277;Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T.,Kosaka, M., Takatuski, K., Yamaya, T., Toyama K., Yoshida, T., Masaoka,T., Hashimoto, S., Ohshima, T., Kimura, I., Yamada, K. and Kimura, J.(1991) “Treatment of myelodysplastic syndromes with orally administered1-β-D-arabinouranosylcytosine -5′ stearylphosphate.” Oncology 48,451–455. Palomino, E., Kessle, D. and Horwitz, J. P. (1989) “Adihydropyridine carrier system for sustained delivery of 2′,3′dideoxynucleosides to the brain.” J. Med. Chem. 32, 22–625; Perkins, R.M., Barney, S. Wittrock, R., Clark, P. H., Levin, R. Lambert, D. M.,Petteway, S. R., Serafinowska, H. T., Bailey, S. M., Jackson, S.,Harnden, M. R. Ashton, R., Sutton, D., Harvey, J. J. and Brown, A. G.(1993) “Activity of BRL47923 and its oral prodrug, SB203657A against arauscher murine leukemia virus infection in mice.” Antiviral Res. 20(Suppl. 1). 84; Piantadosi, C., Marasco, C. J., Jr., Norris-Natschke, S.L., Meyer, K. L., Gumus, F., Surles, J. R., lshaq, K. S., Kucera, L. S.Iyer, N., Wallen, C. A., Piantadosi, S. and Modest, E. J. (1991)“Synthesis and evaluation of novel ether lipid nucleoside conjugates foranti-HIV-1 activity.” J. Med. Chem. 34, 1408–1414; Pompon, A., Lefebvre,I., Imbach, J. L., Kahn, S. and Farquhar, D. (1994). “Decompositionpathways of the mono- and bis(pivaloyloxymethyl) esters ofazidothymidine-5′-monophosphate in cell extract and in tissue culturemedium; an application of the ‘on-line ISRP-cleaning HPLC technique.”Antiviral Chem Chemother. 5, 91–98; Postemark, T. (1974) “Cyclic AMP andcyclic GMP.” Annu. Rev. Pharmacol. 14, 23–33; Prisbe, E. J., Martin, J.C. M., McGhee, D. P. C., Barker, M. F., Smee, D. F. Duke, A. E.,Matthews, T. R. and Verheyden, J. P. J. (1986) “Synthesis and antiherpesvirus activity of phosphate an phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy)methyl]guanine. ” J. Med. Chem. 29, 671–675;Pucch, F., Gosselin, G., Lefebvre, I., Pompon, a., Aubertin, A. M. Dim,and Imbach, J. L. (1993) “Intracellular delivery of nucleosidemonophosphate through a reductase-mediated activation process.”Antiviral Res. 22, 155–174; Pugaeva, V. P., Klochkeva, S. I., Mashbits,F. D. and Eizengart, R. S. (1969). “Toxicological assessment and healthstandard ratings for ethylene sulfide in the industrial atmosphere.”Gig. Trf. Prof. Zabol. 14, 47–48 (Chem. Abstr. 72, 212); Robins, R. K.(1984) “The potential of nucleotide analogs as inhibitors of Retroviruses and tumors.” Pharm. Res. 11–18; Rosowsky, A., Kim. S. H., Rossand J. Wick, M. M. (1982) “Lipophilic 5′-(alkylphosphate) esters of1-β-D-arabinofiiranosylcytosine and its N⁴-acyl and2.2′-anhydro-3′-O-acyl derivatives as potential prodrugs.” J. Med. Chem.25, 171–178; Ross, W. (1961) “Increased sensitivity of the walkerturnout towards aromatic nitrogen mustards carrying basic side chainsfollowing glucose pretreatment.” Biochem. Pharm. 8, 235–240; Ryu, E. K.,Ross, R. J. Matsushita, T., MacCoss, M., Hong, C. I. and West, C. R.(1982). “Phospholipid-nucleoside conjugates. 3. Synthesis andpreliminary biological evaluation of 1-β-D-arabinofuranosylcytosine5′diphosphate [−], 2-diacylglycerols.” J. Med. Chem. 25, 1322–1329;Saffhill, R. and Hume, W. J. (1986) “The degradation of5-iododeoxyuridine and 5-bromoethoxyuridine by serum from differentsources and its consequences for the use of these compounds forincorporation into DNA.” Chem. Biol. Interact. 57, 347–355; Saneyoshi,M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H.(1980) “Synthetic nucleosides and nucleotides. XVI. Synthesis andbiological evaluations of a series of 1-β-D-arabinofuranosylcytosine5′-alky or arylphosphates.” Chem Pharm. Bull. 28, 2915–2923; Sastry, J.K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1 992) “Membrane-permeable dideoxyuridine5′-monophosphate analogue inhibits human immunodeficiency virusinfection.” Mol. Pharmacol. 41, 441–445; Shaw, J. P., Jones, R. J.Arimilli, M. N., Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) “Oralbioavailability of PMEA from PMEA prodrugs in male Sprague-Dawley rats.”9th Annual AAPS Meeting. San Diego, Calif. (Abstract). Shuto, S., Ueda,S., Imamura, S., Fukukawa, K. Matsuda, A. and Ueda, T. (1987) “A facileone-step synthesis of 5′ phosphatidiylnucleosides by an enzymatictwo-phase reaction.” Tetrahedron Lett. 28, 199–202; Shuto, S. Itoh, H.,Ueda, S., Imamura, S., Kukukawa, K., Tsujino, M., Matsuda, A. and Ueda,T. (1988) Pharm. Bull. 36, 209–217. An example of a useful phosphateprodrug group is the S-acyl-2-thioethyl group, also referred to as“SATE”.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1. A method for treating an HIV infection in a human comprisingadministering an effective amount of β-D-D4FC or its pharmaceuticallyacceptable ester or salt to the human, optionally in a pharmaceuticallyacceptable carrier, in combination or alternation with an effectiveamount of abacavir.
 2. A pharmaceutical composition comprising aneffective amount of β-D-D4FC or its pharmaceutically acceptable ester orsalt, in a pharmaceutically acceptable carrier, in combination with aneffective amount of abacavir.
 3. The method of claim 1, wherein β-D-D4FCand abacavir are administered in a pharmaceutically acceptable carrier.4. The method according to claim 3, wherein the pharmaceuticallyacceptable carrier is suitable for oral delivery, intravenous delivery,parenteral delivery, intradermal delivery, subcutaneous delivery ortopical delivery.
 5. The method of claim 1, wherein β-D-D4FC andabacavir are in the form of a dosage unit.
 6. The method of claim 5,wherein the dosage unit contains 10 to 1500 mg of each compound.
 7. Themethod of claim 5, wherein the dosage unit is a tablet or capsule. 8.The method of claim 1 wherein β-D-D4FC and abacavir are administered incombination.
 9. The method of claim 1 wherein β-D-D4FC and abacavir areadministered in alternation.
 10. The pharmaceutical compositionaccording to claim 2, wherein the β-D-D4FC and abacavir are in apharmaceutically acceptable carrier.
 11. The pharmaceutical compositionaccording to claim 10, wherein the pharmaceutically acceptable carrieris suitable for oral delivery, intravenous delivery, parenteraldelivery, intradermal delivery, subcutaneous delivery or topicaldelivery.
 12. The pharmaceutical composition according to claim 11,wherein the pharmaceutically acceptable carrier is suitable for oraldelivery.
 13. The pharmaceutical composition according to claim 11,wherein the pharmaceutically acceptable carrier is suitable forintravenous delivery.
 14. The pharmaceutical composition according toclaim 11, wherein the pharmaceutically acceptable carrier is suitablefor parenteral delivery.
 15. The pharmaceutical composition according toclaim 11, wherein the pharmaceutically acceptable carrier is suitablefor intradermal delivery.
 16. The pharmaceutical composition accordingto claim 11, wherein the pharmaceutically acceptable carrier is suitablefor topical delivery.
 17. The pharmaceutical composition according toclaim 2, wherein β-D-D4FC and abacavir are in the form of a dosage unit.18. The pharmaceutical composition according to claim 17, wherein thedosage unit contains 10 to 1500 mg of each compound.
 19. Thepharmaceutical composition according to claim 17, wherein the dosageunit is a tablet or capsule.